Androgen regulates VEGF expression in LNCaP and 22Rv1 prostate cancer cells. (A) 22Rv1 cells were serum starved overnight then treated with 5nM R1881 or DMSO as a vehicle control (0nM R1881). VEGF mRNA expression was measured by qRT-PCR and normalized by β-actin levels as described in text. (B) VEGF mRNA expression in LNCaP cells was measured by qRT-PCR and normalized by 18S levels, as described in text. Cells were serum starved as described in A. For inhibition of androgen activity, cells were pre-treated with 10μM casodex for 2 hrs and then induced with 5nM R1881 for 24 hrs. Values represent fold change relative to DMSO treatment. A Student’s t-test was performed and significance was determined * (p < 0.05), ** (p < 0.01). (C) VEGF protein expression in LNCaP cells treated with 1nM R1881 for 0–48 hours. Protein expression was measured by western blotting as described in text, and β-actin levels were used as loading controls. (D) Cytoplasmic VEGF protein expression was measured by western blot of LNCaP cells treated as per (B). Image J analysis was performed and VEGF levels were normalized to β-actin levels. Shown are relative fold-changes in VEGF protein levels, normalized to β-actin and relative to untreated cells.